Biosurfactants are surface active agents produced by microorganisms and are known to possess various bioactivities as well as surface activity. Since biosurfactants exhibit a high degree of biodegradability and are low toxic and hypoallergenic to animals and plants, these environmentally and human friendly compounds are expected to be applied in various fields. There have been known some biosurfactants such as glycolipid biosurfactants, peptide biosurfactants, and fatty acid biosurfactants.
Sophorose lipids are a type of glycolipid biosurfactants and have a structure in which sophorose consisting of two glucose molecules is linked to a fatty acid by a glycosidic ether bond. Sophorose lipids are categorized into two forms: the lactone form in which the carboxyl group in the fatty acid side chain and the sophorose moiety form a cyclic ester bond; and the acid form in which the bond is hydrolyzed. In addition to these forms, there exists a number of derivatives which are characterized by the presence or absence of double bonds in the fatty acid side chain, the length of the carbon chain, the position of the glycosidic ether bond, the presence or absence of acetyl groups introduced to the hydroxyl groups of the sugar moiety, and other structural parameters (Non-Patent Document 1). Cultivation of yeast cells on a culture substrate including a sugar and/or an oil or fat provides a mixture of these forms of sophorose lipid, and the composition of the mixture depends on the used culture substrate (Non-Patent Document 2). The composition of the fatty acid side chains of the sophorose lipids also depends on the type of culture substrate used (Non-Patent Document 2).
As known in the art, sophorose lipids are generally produced in a highly viscous oil form that is difficult to handle (Non-Patent Document 3). For example, a sophorose lipid oil obtained from a liquid culture by inclined sedimentation is an oil with a water content of 40 to 50%. Then, sophorose lipids are extracted from this oil with an organic solvent in order to increase the purity, and the obtained sophorose lipids have a remarkably high viscosity (Patent Document 1).
The use of a culture substrate including an alkane such as hexadecane results in the production of sophorose lipids mainly composed in particular of the diacetyl lactone form among the lactone forms. The diacetyl lactone form is known to have comparatively high hydrophobicity and crystallize out of the liquid culture (Non-Patent Document 4). This method is useful because sophorose lipids are produced in an easy-to-handle solid form. However, industrial application of this method is impractical because alkanes to be used are expensive materials.
Many studies on the production of sophorose lipids using an inexpensive material such as a vegetable oil or fat, or a fatty acid derived from a vegetable oil or fat have been reported. However, all of them demonstrate the production of sophorose lipids in an oil form, not in a solid form, except only one that demonstrates the production of sophorose lipids in a solid form from a liquid culture obtained by using a culture substrate including Turkish corn oil (Non-Patent Document 5). Non-Patent Document 5, however, teaches that sophorose lipids may be produced in a viscous honey form depending on the culture method. In other words, Non-Patent Document 5 fails to provide sufficient knowledge of the culture conditions for producing solid sophorose lipids. In addition, Non-Patent Document 5 does not teach any composition ratio of the produced solid sophorose lipids (e.g. the ratio of the lactonic form and the acid form, the ratio of the diacetyl lactone form, the monoacetyl lactone form and the diol lactone form, and the ratio of saturated fatty acid side chains to unsaturated fatty acid side chains in all fatty acid chains).
There are also many studies on improvement of the productivity of sophorose lipids. It is known that a key to increase the production of sophorose lipids is to culture microorganism cells in aerobic conditions (Non-Patent Document 6). One measure to achieve this culture conditions is, for example, to increase the stirrer speed to maintain a high dissolved oxygen saturation level when the dissolved oxygen saturation level decreases with proliferation of the microorganism cells (Non-Patent Document 5). All of these studies also do not teach any relationship between culture conditions (e.g. aeration conditions) and the composition ratio of produced sophorose lipids (e.g. the ratio of the lactone form and the acid form, the ratio of the diacetyl lactone form, the monoacetyl lactone form and the diol lactone form, and the ratio of saturated fatty acid side chains to unsaturated fatty acid side chains in all fatty acid chains).
Lactonic sophorose lipids possess stronger antibacterial activities compared to acidic sophorose lipids, and in particular, sophorose lipids in the diacetyl form or the monoacetyl form are known to possess further stronger activities (Non-Patent Document 7). In addition, lactonic sophorose lipids, for example, obtained from a liquid culture obtained by using a culture medium including oleic acid have been reported to possess antibacterial and antifungal activities (Patent Documents 2 and 3 and Non-Patent Document 7). All of these reports, however, do not teach any relationship between the structure of the fatty acid side chain of the sophorose lipid and its antibacterial activity.
Acidic sophorose lipids are highly water soluble. In the case that acidic sophorose lipids are produced in a liquid culture, isolation thereof from the culture is difficult. Only purification by chromatography is known to do this (Non-Patent Document 1), but it is substantially impractical for industrial applications. A method for producing acidic sophorose lipids by hydrolyzing lactonic sophorose lipids is also known (Patent Document 1). However, at present, it is remarkably difficult to obtain high purity acidic sophorose lipids at low cost because, in the first place, there is no known method for obtaining high purity lactonic sophorose lipids at low cost.    Patent Document 1: JP-A S54-28895    Patent Document 2: WO 2004/044216    Patent Document 3: WO 2006/069175    Non-Patent Document 1: Journal of the American Oil Chemists' Society, vol. 65, no. 9, 1460 (1988)    Non-Patent Document 2: Journal of Industrial Microbiology, vol. 13, 249 (1994)    Non-Patent Document 3: Canadian Journal of Chemistry, vol. 39, 846 (1961)    Non-Patent Document 4: Journal of the American Oil Chemists' Society, vol. 83, no. 2, 137 (2006)    Non-Patent Document 5: Engineering in Life Sciences, vol. 5, no. 4, 357 (2005)    Non-Patent Document 6: Applied Microbiology and Biotechnology, vol. 76, 23 (2007)    Non-Patent Document 7: Fett Wissenschaft Technologie, vol. 91, 363 (1989)